Frequent recombination contributes significantly to the diversity of the HIV-1 population. We have studied multiple aspects of HIV-1 recombination, including the mechanisms that generate intersubtype recombinants, which are playing an increasingly important role in the current AIDS epidemic. By comparing intra- and intersubtype HIV-1 recombination, we have found that the sequence diversity between different HIV-1 subtypes decreases the crossover events and reduces the replication fitness of the recombinants, thereby causing the loss of newly generated chimeric viruses. Additionally, the dimerization initiation signal (DIS), a 6-nt palindromic sequence in the 5? untranslated region of the viral genome, affects the HIV-1 recombination frequency by two separate mechanisms: first, the identity of the DIS affects the generation of recombinants between genotypes with different DIS by dictating the frequency of viral RNA copackaging; second, discordant DIS sequences in the copackaged RNAs can further decrease crossovers at the 5? end of the viral genome and generate a recombination gradient. As HIV-1 is thought to be a recombinant generated from two distinct primate lentiviruses, we also studied recombination between different AIDS viruses. We demonstrated that recombination can occur between distantly related HIV-1 and HIV-2, as well as group O and group M HIV-1 variants, albeit at low rates. These studies revealed insights into the recombination mechanisms that generate diversity in the HIV-1 genome and potentially novel chimeric viruses. Our future efforts in this project will be focused on two subaims. We will study a critical step of reverse transcription, minus-strand DNA transfer, during which DNA synthesis switches from using one end of the RNA template to the other end. We will use genetic assays to probe the mechanisms of this process, including whether the template can be in an alternative form that brings the distance between the two ends close together. We will also study mechanisms that cause a loss of replication fitness in newly generated intersubtype recombinants. These studies will reveal insights into the replication mechanisms of HIV-1.[Corresponds to Hu Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]